High-Throughput Platform for Detection of Neutralizing Antibodies Using Flavivirus Reporter Replicon Particles.

Flavivirus outbreaks require fast and reliable diagnostics that can be easily adapted to newly emerging and re-emerging flaviviruses. Due to the serological cross-reactivity among flavivirus antibodies, neutralization tests (NT) are considered the gold standard for sero-diagnostics. Here, we first established wild-type single-round infectious virus replicon particles (VRPs) by packaging a yellow fever virus (YFV) replicon expressing Gaussia luciferase (Gluc) with YFV structural proteins in trans using a double subgenomic Sindbis virus (SINV) replicon. The latter expressed the YFV envelope proteins prME via the first SINV subgenomic promoter and the capsid protein via a second subgenomic SINV promoter. VRPs were produced upon co-electroporation of replicon and packaging RNA. Introduction of single restriction enzyme sites in the packaging construct flanking the prME sequence easily allowed to exchange the prME moiety resulting in chimeric VRPs that have the surface proteins of other flaviviruses including dengue virus 1--4, Zika virus, West Nile virus, and tick-borne encephalitis virus. Besides comparing the YF-VRP based NT assay to a YF reporter virus NT assay, we analyzed the neutralization efficiencies of different human anti-flavivirus sera or a monoclonal antibody against all established VRPs. The assays were performed in a 96-well high-throughput format setting with Gluc as readout in comparison to classical plaque reduction NTs indicating that the VRP-based NT assays are suitable for high-throughput analyses of neutralizing flavivirus antibodies.

Characterization and Vector Competence Studies of Chikungunya Virus Lacking Repetitive Motifs in the 3' Untranslated Region of the Genome.

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3' untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.

Seroepidemiology of Chikungunya Virus Among Febrile Patients in Eight Health Facilities in Central and Northern Mozambique, 2015-2016.

BACKGROUND: The burden and spread of chikungunya virus (CHIKV) are rapidly increasing worldwide, but the epidemiology in Mozambique is barely known. The aim of this study was to determine the seroepidemiology of CHIKV in central and northern Mozambique. METHODS: A cross-sectional study was conducted between March 2015 and May 2016 in eight health facilities situated in central and northern Mozambique to recruit 392 patients with undifferentiated febrile illness from outpatient clinics. Serum samples from each participant were screened using commercially available enzyme-linked immunosorbent assay for detection of anti-CHIK IgM and IgG antibodies. A subset of study samples (n = 37) was further tested by the plaque reduction neutralization assay (PRNT). RESULTS: The median age of participants was 23 years (IQR: 7-34), and 45.7% were female. The frequency of participants with seropositivity for IgM and IgG anti-CHIKV antibodies was 1.5% (6/392) and 28.6% (112/392), respectively. Patients with seropositivity for IgM anti-CHIKV were significantly younger. Frequency of patients with seropositivity for IgG anti-CHIKV increased with age. Frequency of patients with seropositivity for IgM anti-CHIKV was higher in Tete province, but most patients with seropositivity for IgG anti-CHIKV infection were from Cabo Delgado and Sofala provinces. CONCLUSIONS: Our data demonstrate serological evidence of CHIKV in central and northern Mozambique, expanding the limited evidence of the virus in the country. We recommend that CHIKV should be considered in the differential diagnosis of febrile illness throughout the country.

Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle.

The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d'Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release. IMPORTANCE Most viruses of the genus Flavivirus, e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers.

A basic cluster in the N terminus of yellow fever virus NS2A contributes to infectious particle production.

UNLABELLED: The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2Aα cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. IMPORTANCE: Despite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of amino acids in the N terminus of YFV NS2A which inhibited virion assembly upon mutation. The defect was rescued by a spontaneously occurring mutation in NS3. Our study proves an interaction between NS2A and NS3, which, remarkably, was maintained for the NS2A mutant in the presence and absence of the NS3 mutation. This suggests a role for other viral and/or cellular proteins in virion assembly. Residues important for YFV virion production reported here only partially coincided with those reported for other flaviviruses, suggesting that the determinants for particle production are virus specific. Reconstruction of a YFV encoding tagged NS2A paves the way to identify further NS2A interaction partners.

Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites.

Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research.

Full genome sequence analysis of parechoviruses from Brazil reveals geographical patterns in the evolution of non-structural genes and intratypic recombination in the capsid region.

Due to high genome plasticity, the evolutionary fate and geographical history of picornaviruses is hard to follow. Here, we determined the complete coding sequences of eight human parechoviruses (HPeV) of types 1, 5 and 6 directly from clinical samples from Brazil. The capsid genes of these strains were not remarkably different from European, North American and Japanese HPeV. Full genome analysis revealed frequent intertypic recombination in the non-structural genome region. In addition, evidence of recombination between viruses of the same type in the capsid-encoding genome region among HPeV1 and HPeV4 was obtained. Bayesian phylogenetic analysis indicated that strains without evidence of recombination with each other in any genome region were separated by no more than 35 years of circulation. Interestingly, in the 3C gene, all Brazilian parechoviruses grouped together regardless of serotype. The most recent common ancestor of these strains dated back 108 years, suggesting long-term endemicity of this particular P3 genome lineage in South America. Our results support the idea that picornavirus replicative genes acquire capsid proteins introduced by new strains. Under certain epidemiological conditions, replicative genes may be maintained in circumscript geographical regions.